Adenovirus (Ad) is a linear double-stranded DNA virus. Successful lytic infection by Ad requires a productive interaction with its host cell, creating an environment that is conducive to efficient viral gene expression, replication, and assembly of new progeny virus. Ad regulatory proteins derived from early region 4 (E4), E4-11K and E4-34K, are important in cell transformation, viral replication, and late gene expression; they are also able to interact with host double-strand break repair (DSBR) proteins and affect their localization and stability. E4 mutants have their genomes "repaired" in infected cells to form concatemers of several genome lengths. Here, we propose to investigate the role of host DSBR processes in the replication and late gene expression defects displayed by E4 mutants lacking E4-11K and E4-34K. The experiments will investigate the relationship between the function of E4 proteins in inactivating DSBR, and their role in promoting viral replication and gene expression. We will study the significance of genome concatenation for viral late gene expression in E4 mutant infections. We will study the role of individual DSBR proteins and genome concatenation in the onset of E4 mutant DNA replication. The ability of Ad genomes to induce a damage response in the presence and absence of viral DNA replication will be assessed. Finally, we will study the role of nuclear domain 10 (ND10) disruptions for viral replication. ND10 contains DSBR proteins that are targeted by E4 regulatory proteins. Our goals are to investigate possible links between the ability of E4 proteins to inactivate host DSBR, and their function in promoting viral replication and gene expression during lytic infections. The project has long term significance for understanding how viral genes affect host cell activities, and how they may ultimately affect cell growth and transformation as they reprogram the machinery of the host to promote a successful lytic viral infection.